Pancreatic ductal adenocarcinoma (PDAC) is an aggressive and poorly understood disease that has the worst prognosis of all cancers (5 year survival of 7%), with more than 40,000 deaths per year in the US. Standard- of-care treatment for PDAC includes surgery and chemotherapy. Many patients receive chemotherapy after surgery (?adjuvant treatment?), to improve progression-free survival. About 45-55% of patients present with metastatic disease and immediately begin chemotherapy if tolerated. Unfortunately, there are significant toxicities associated with chemotherapy, and there are no tools to determine whether a patient will benefit from a more toxic versus a less toxic drug. Additionally, there is no rational system to match each patient with the most promising new drug for their cancer. Finally, there are few effective drugs for PDAC, and therefore a demand for accelerated drug development. Thus, there is a critical need to improve the care of PDAC patients through reduced toxicities, rational treatment planning, and new drug development. The goal of this proposal is to develop novel cellular-level imaging technologies to predict treatment response in individual PDAC patients, using macro-suspensions of the patients? tumors maintained in a 3D culture (?organoids?). This non-invasive optical metabolic imaging (OMI) approach exploits the intrinsic fluorescence intensity and lifetime of the metabolic co-enzymes NADH and FAD to image drug response on a single-cell level across all cells in the intact 3D sample. This single-cell analysis allows for heterogeneous drug response to be monitored over a treatment time-course in tumor cells and stromal cells. This is important for assessing treatment efficacy in the highly heterogeneous and stromal micro-environment of PDAC. The use of primary tumor organoids derived from the patients? own tumor also maintains the cell-cell communication, 3D architecture, and tumor-stromal interactions that are critical to accurately assess drug response. Our preliminary data indicate that OMI in primary PDAC organoids (1) accurately predicts in vivo drug response in mice, and (2) can identify drugs that effectively target tumor fibroblasts for stromal re-organization and improved drug delivery. We have also established feasibility for human testing in 6 PDAC patients. This novel platform provides great potential for (1) rapidly testing new drug regimens on relevant patient samples, thus accelerating PDAC drug development, and (2) providing individualized drug screens to identify the most effective and least toxic treatment for each patient. This proposal will test the hypothesis that OMI of primary PDAC organoids can accurately predict in vivo treatment efficacy in mice and humans. This approach will be validated on mouse models of PDAC, on primary patient samples in the adjuvant treatment setting, and on primary patient samples in the metastatic treatment setting. The proposed development of dynamic, single-cell assessment techniques to monitor drug response in multiple cell types within intact mouse and human PDAC organoids holds great promise for rational drug development and treatment planning that could ultimately reduce toxicities and improve outcomes in patients.